Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula

Schefe, Jan H.; Lehmann, Kerstin; Buschmann, Ivo; Unger, Thomas; Funke-Kaiser, Heiko

doi:10.1007/s00109-006-0097-6

Cited by 752 publications

(549 citation statements)

References 31 publications

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“…The efficiency of every PCR reaction was calculated from the slope of its linear amplication region using at least 4 data points with LinRegPCR software (Ramakers et al, 2003;Supplemental Table 1). Efficiencies were either the same or the largest average difference was 0.02 (see Supplemental Table 1) with standard deviations of <= 0.02, the standard stated by Schefe et al (2006). The correlations (R-square) for each individual efficiency was >= 0.998, demonstrating a good fit.…”

Section: Discussionmentioning

confidence: 87%

Motor deficits and altered striatal gene expression in aphakia (ak) mice

et al. 2007

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Like humans with Parkinsons disease (PD), the ak mouse lacks the majority of the substantia nigra pars compacta (SNc) and experiences striatal denervation. The purpose of this study was to test whether motor abnormalities in the ak mouse progress over time, and whether motor function could be associated with temporal alterations in the striatal transcriptome. Ak and wt mice (28 to 180 days old) were tested using paradigms sensitive to nigrostriatal dysfunction. Results were analyzed using a linear mixed model. Ak mice significantly underperformed wt controls in rotarod, balance beam, string test, pole test and cotton shred tests at all ages examined. Motor performance in ak mice remained constant over the first 6 months of life, with the exception of the cotton shred test, in which ak mice exhibited marginal decline in performance. Dorsal striatal semi-quantitative RT-PCR for 19 dopaminergic, cholinergic, glutaminergic and catabolic genes was performed in 1 and 6 month old groups of ak and wt mice. Preproenkephalin levels in ak mice were elevated in both age groups. Drd1, 3 and 4 levels declined over time, in contrast to increasing Drd2 expression. Additional findings included decreased Chrnα6 expression and elevated VGluT1 expression at both time points in ak mice, and elevated AchE expression in young ak mice only. Results confirm that motor ability does not decline significantly for the first 6 months of life in ak mice. Their striatal gene expression patterns are consistent with dopaminergic denervation, and change over time, despite relatively unaltered motor performance.

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Section: Discussionmentioning

confidence: 87%

Motor deficits and altered striatal gene expression in aphakia (ak) mice

et al. 2007

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“…Duplicate 25-μl real-time polymerase chain reaction (PCR) reactions were performed in 96-well plates using a SYBR-Green reaction mix and a Biorad iCycler according to the supplier's protocol with the following forward and reverse primers: glyceraldehydes-3 phosphate dehydrogenase (GAPDH), 5′-agcctcgtcccgtaga caaaat and 5′-tggcaacaatctccactttgc and HSP72, 5′-accaag cagacgcagatcttc and 5′-agcctcaagatcatcagcaatg. Data were quantified using the gene expression Ct difference method described by Schefe et al (2006) and standardized to levels of the housekeeping gene, GAPDH using Ct values automatically determined by the thermocycler. Briefly, the efficiency of amplification for each primer pair was calculated using the equation:…”

Section: Methodsmentioning

confidence: 99%

Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells

Tulapurkar

Asiegbu

Singh

et al. 2009

Cell Stress and Chaperones

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Expression of heat shock proteins (HSPs) is classically activated at temperatures above the physiologic range (≥42°C) via activation of the stress-activated transcription factor, heat shock factor-1 (HSF-1). Several studies suggest that less extreme hyperthermia, especially within the febrile range, as occurs during fever and exertional/environmental hyperthemia, can also activate HSF-1 and enhance HSP expression. We compared HSP72 protein and mRNA expression in human A549 lung epithelial cells continuously exposed to 38.5°C, 39.5°C, or 41°C or exposed to a classic heat shock (42°C for 2 h). We found that expression of HSP72 protein and mRNA increased linearly as incubation temperature was increased from 37°C to 41°C, but increased abruptly when the incubation temperature was raised to 42°C. A similar response in luciferase activity was observed using A549 cells stably transfected with an HSF-1-responsive luciferase reporter plasmid. However, activation of intranuclear HSF-1 DNA-binding activity was comparable at 38.5°C, 39.5°C, and 41°C and only modestly greater at 42°C but the mobility of HSF1 protein on a denaturing gel was altered with increasing exposure temperature and was distinctly different at 42°C. These findings indicate that the proportional changes in HSF-1-dependent HSP72 expression at febrile-range temperatures are dependent upon exposure time and temperature but not on the degree of HSF-1 DNA-binding activity. Instead, HSF-1-mediated HSP expression following hyperthermia and heat shock appears to be mediated, in addition to HSF-1 activation, by posttranslational modifications of HSF-1 protein.

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“…Each cDNA was analyzed in quadruplicate, and the average threshold cycle (Ct) was calculated for each sample. The relative expression levels were calculated using the 2 -△△CT method, 25) and the average Ct was analyzed for all genes to correct for differences in the cDNA input. All reactions were performed in duplicate.…”

Section: Rt-qpcrmentioning

confidence: 99%